Transcriptional Repression by FACT Is Linked to Regulation of Chromatin Accessibility at the Promoter of ES Cells

Published Online: 13 June, 2018 | Supp Info: http://doi.org/10.26508/lsa.201800085 Downloaded from life-science-alliance.org on 26 September, 2021 Research Article Transcriptional repression by FACT is linked to regulation of chromatin accessibility at the promoter of ES cells Constantine Mylonas1 , Peter Tessarz1,2 The conserved and essential histone chaperone, facilitates chro- the yeast homolog Pob3. In yeast, an HMG domain protein named matin transcription (FACT), reorganizes nucleosomes during DNA Nhp6 has been proposed to provide the DNA binding capacity of transcription, replication, and repair and ensures both efficient FACT (Formosa et al, 2001). elongation of RNA Pol II and nucleosome integrity. In mammalian The molecular basis for FACT activity has long remained elusive. cells, FACT is a heterodimer, consisting of SSRP1 and SUPT16. Here, However, recent biochemical and structural studies are starting to we show that in contrast to yeast, FACT accumulates at the tran- elucidate how FACT engages nucleosomes (Winkler & Luger, 2011; scription start site of genes reminiscent of RNA polymerase II profile. Hondele et al, 2013; Hsieh et al, 2013; Kemble et al, 2015). Via its Depletion of FACT in mouse embryonic stem cells leads to de- several domains, FACT binds to multiple surfaces on the nucleo- regulation of developmental and pro-proliferative genes concom- some octamer and acts by shielding histone–DNA interactions. itant with hyper-proliferation of mES cells. Using MNase-seq, Assay Initially, it was proposed that FACT would evict an H2A/B dimer from for Transposase-Accessible Chromatin sequencing, and nascent the nucleosome in front of the polymerase and then reinstate elongating transcript sequencing, we show that up-regulation of nucleosome integrity in its wake. However, other data suggest that genes coincides with loss of nucleosomes upstream of the tran- this dimer replacement is not part of FACT function as it leaves the scription start site and concomitant increase in antisense tran- histone composition of the nucleosome intact (Formosa, 2012). scription, indicating that FACT impacts the promoter architecture to Based on recent biochemical data (Hsieh et al, 2013), a model regulate the expression of these genes. Finally, we demonstrate emerges in which RNA Pol II enters the nucleosome and partially a role for FACT in cell fate determination and show that FACT de- uncoils the nucleosomal DNA. At the same time, FACT binds to the pletion primes embryonic stem cells for the neuronal lineage. proximal and distal H2A/H2B dimer, and these FACT–dimer interactions facilitate nucleosome survival. DOI 10.26508/lsa.201800085 | Received 8 May 2018 | Revised 29 May 2018 | Accepted 29 May 2018 | Published online 13 June 2018 Although the genetics and biochemistry of FACT are relatively well understood, it is not known whether cell-type dedicated functions are conferred by this histone chaperone. Interestingly, genome-wide expression analyses across cell and tissue types implicate a role of Introduction FACT in maintaining an undifferentiated state. Depletion of FACT subunits leads to growth reduction in transformed but not in im- The basic functional unit of chromatin is the nucleosome consisting mortalized cells (Garcia et al, 2013), indicating that FACT is essential of around 147 bp of DNA wrapped around an octamer of histone for tumour growth but not for proliferation of untransformed cells. proteins—two copies each of histones H2A, H2B, H3, and H4. In vitro, Finally, FACT regulates the expression of Wnt target genes during chromatinized DNA templates are refractory to transcription, osteoblast differentiation in mesenchymal stem cells and its de- suggesting that the nucleosome might provide a barrier for the letion leads to a differentiation skew (Hossan et al, 2016). Taken elongating RNA polymerase. Using elegant biochemical fraction- together, these data suggested a more specific role for the FACT ation assays coupled to in vitro transcription assays, facilitates complex in undifferentiated cells as previously assumed. chromatin transcription (FACT) was initially characterised as Recent studies have demonstrated that RNA Pol II can transcribe a factor that alleviated the repressive nature of chromatin in vitro in both sense and antisense directions near many mRNA genes (Orphanides et al, 1999). Meanwhile, it has been demonstrated that (Kwak et al, 2013; Mayer et al, 2015). At these so-called bidirectional FACT can cooperate with all RNA polymerases in the cell and ensure promoters, RNA Pol II initiates transcription and undergoes both efficient transcription elongation and nucleosome integrity. promoter-proximal pausing in both the sense (at the protein- Both FACT subunits are highly conserved across all eukaryotes with coding transcription start site [TSS]) and antisense orientation the exception of an HMG-like domain present in SSRP1 but absent in (Kwak et al, 2013; Mayer et al, 2015). Divergent transcription is often 1Max Planck Institute for Biology of Ageing, Cologne, Germany 2Cologne Excellence Cluster on Cellular Stress Responses in Ageing Associated Diseases, University of Cologne, Cologne, Germany Correspondence: [email protected] ©2018Tessarz and Mylonas https://doi.org/10.26508/lsa.201800085 vol 1 | no 3 | e201800085 1of14 found at mammalian promoters that are rich in CpG content, but actively transcribed genes (Carvalho et al, 2013). We suspect that the lack key core promoter elements such as the TATA motif (Scruggs strong enrichment of FACT subunits around the TSS might mask this et al, 2015). A broader nucleosome free region (NFR) in the promoter potential correlation. Nevertheless, FACT subunits also co-localize region is often accompanied by divergent transcription, and can to the gene body of actively transcribed genes and enrich towards lead to binding of more transcription factors (TFs) resulting in the transcription end site, similarly to H3K36me3 (Fig S1C and D). higher gene activity (Scruggs et al, 2015). Pearson’s correlation among FACT and active marks remained el- Here, we have confirmed an indispensable role of FACT in un- evated when we focused on promoter and enhancer regions (n = differentiated cells based on the expression levels of both FACT 19,461) (Fig 1B). Both subunits displayed very similar binding pattern subunits and, thus, chose mouse embryonic stem cells as a model to to each other over the TSS of all the annotated genes and were investigate how FACT might shape the transcriptome and maintain tightly linked to H3K4me3 levels (Fig 1C). an undifferentiated state. To achieve this, we performed chromatin immunoprecipitation and sequencing (ChIP-seq) and RNA-seq Regulation of gene expression by FACT to identify genes bound and regulated by FACT. To address at a mechanistic level how FACT might regulate transcription in embry- To investigate how FACT orchestrates transcriptional regulation in onic stem (ES) cells, we combined this analysis with MNase digestion mESCs, we depleted SSRP1 levels using short hairpin RNAs (shRNA; Fig of chromatin coupled to deep sequencing (MNase-seq), assay for S2A). Importantly, this also led to a simultaneous depletion of SUPT16 transposase-accessible chromatin using sequencing (ATAC-seq), levels as assessed by mass spectrometry (Table S6). This in- and nascent elongating transcript sequencing (NET-seq). Using terdependence of the two FACT subunits has been observed before these approaches, we have identified a specific gene cluster com- (Garcia et al, 2013). Surprisingly, we observed an increase in mESC prising genes involved in embryogenesis/neuronal development proliferation following Ssrp1 knockdown (KD) as measured by pro- that are up-regulated upon FACT depletion. In addition, we observed liferation rate via metabolic activity measurement (MTT) cell pro- a concomitant increase in chromatin accessibility around the TSS, liferation assays using independent shRNAs (Figs 2A and S2B). This is suggesting that maintenance of nucleosomes at this position by FACT in contrast to previously published data from tumour cell lines, in is part of the mechanism how FACT impacts on the regulation of which proliferation rates decrease, and also from terminally differ- these genes. Finally, our data support a role of FACT in the main- entiated cells, where FACT depletion has no effect on proliferation tenance of a pluripotent state by showing that its depletion leads to (Garcia et al, 2013). Subsequently, we sequenced the whole tran- faster differentiation into the neuronal lineage. scriptome (RNA-seq). In total, we characterized 3,003 differentially expressed genes: 1,655 down-regulated and 1,348 up-regulated (Fig 2B). Down-regulated genes were overrepresented for pathways involved in development, whereas up-regulated genes were involved in Results metabolic processes and positive regulation of proliferation (Fig 2C), indicating that the change in the transcriptome accounts for the faster Occupancy of FACT correlates with marks of active gene proliferation rates. These results suggest that FACT impacts de- expression velopmental processes and negatively controls cell proliferation in mES cells by controlling gene expression patterns. A low correlation High expression of FACT has been associated with stem or less- (Pearson’s R = 0.11) was observed between the coverage of SSRP1 differentiated cells (Garcia et al, 2011). Indeed, we were able to (ChIP-seq) and the gene fold change (RNA-seq) of those genes in the confirm that low FACT levels correlate with highly differentiated

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